To assess the number of dead cells, CAD cells were double stained with PI (catalog number D1306, Molecular Probes, USA) and Hoechst 33342 (catalog number P1304MP, Sigma-Aldrich, USA), which exhibited a red and blue uorescence, respectively. PI is used to count dead cells because it is unable to penetrate viable cells, but can label dead cells. Hoechst was used in order to count the total numbers of cells. The uorescence signals were detected using an Operetta automated wide eld microscope (PerkinElmer, USA). Five non-overlapping elds from each well were randomly selected for imaging. Using the Image J software (RRID:SCR_003070), the percentage of dead cells was calculated as the fraction of PI-positive cells relative to the total cell count.
Operetta automated wide eld microscope
Manufactured by PerkinElmer
Sourced in United States
The Operetta automated wide field microscope is a high-performance imaging system designed for advanced cell-based assays. It features automated acquisition, analysis, and data management capabilities to support a wide range of applications in life science research and drug discovery.
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2 protocols using operetta automated wide eld microscope
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Quantifying Dead Cells via Fluorescence
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Quantifying Dead Cells via Fluorescence
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To assess the number of dead cells, CAD cells were double stained with PI (catalog number D1306, Molecular Probes, USA) and Hoechst 33342 (catalog number P1304MP, Sigma-Aldrich, USA), which exhibited a red and blue uorescence, respectively. PI is used to count dead cells because it is unable to penetrate viable cells, but can label dead cells. Hoechst was used in order to count the total numbers of cells. The uorescence signals were detected using an Operetta automated wide eld microscope (PerkinElmer, USA). Five non-overlapping elds from each well were randomly selected for imaging. Using the Image J software (RRID:SCR_003070), the percentage of dead cells was calculated as the fraction of PI-positive cells relative to the total cell count.
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